Transcriptomic Profile and Expression of Immune Checkpoints in Peripheral Blood Mononuclear Cells of Women with Breast Fibroadenoma

Background — Fibroepithelial tumors of the mammary gland known as fibroadenoma (FA), being one of the most common variants, represent a rather heterogeneous group of diseases, the pathogenetic mechanisms of which remain largely unknown. This study aimed at analyzing two categories of putative changes in the cell-mediated immunity in women diagnosed with FA: (a) changes affecting the transcriptomic profile of the peripheral blood mononuclear cell (PBMC) fraction and the spectrum of activated/ dysregulated signaling pathways that form a specific immune and stromal environment of the tumor; and (b) changes in (co-)expression of immune checkpoint markers — CD279/PD-1, CD274/PD-L1, CD366/TIM3 and CD223/LAG3 – in the CD4 and CD8 subsets of circulating T lymphocytes. Methods — Transcriptome profiling was performed using high-throughput RNA sequencing on the Illumina next-generation sequencing platform, followed by bioinformatic analysis of the resulting datasets including differential gene expression and signaling pathway enrichment analysis. Immune checkpoint marker expression patterns were identified using multicolor flow cytometry. Results — Principal component analysis and hierarchical clustering of the transcriptome datasets revealed three clusters, with the FA cluster clearly distinguishable from the control group. DESeq2 software package detected a total of 334 genes with differential expression between PBMCs from FA patients and healthy controls (with an adjusted p-value 2.0). Gene Ontology (GO) and KEGG enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in over 500 GO terms of the Biological Processes sub-ontology with a false discovery rate (FDR) set to